Chromatography is a widely used method for analyzing and purifying mixtures of compounds from diverse sources such as natural products, combinatorial libraries, pharmaceutical agents and derivatives thereof, and nucleic acids. Purification and analysis of nucleic acids in particular poses challenging resolution difficulties due to the amphiphilic nature of nucleic acid molecules.
Further, synthesis of oligonucleotides requires fast and reliable purification methods in order to increase the speed and efficiency of the synthesis of the full length products. Synthesis of longer oligonucleotides adds considerably to the challenges in purification and synthetic efficiency. A decrease in the time and expense for purification of products is desired.
Current oligonucleotide purification technologies are adapted to purification of “trityl on” oligonucleotides or “trityl off” oligonucleotides, typically involving use of a reversed phase separation step, utilizing the hydrophobicity of the trityl tag to remove failure sequences and impurities. Short oligonucleotides (generally less than 40 bases) can be purified using reversed phase HPLC using this method, and then can be detritylated and desalted. However, there is a need for efficient separation of “trityl on” from “trityl off” oligonucleotides in order to obtain the full length synthetic product, uncontaminated with failure sequences.
The use of “long oligonucleotides,” longer than a 50 mer (i.e., having 50 or more bases or base pairs), is becoming more prevalent and desirable because the longer oligonucleotides are ideal candidates for both microarrays and gene synthesis applications. Kane, M. D., et al., (2000) Nucleic Acids Res. 28, 4552–4557. However, current techniques for purification, for example high performance liquid chromatography (HPLC) and cartridge purification, are not effective for longer oligonucleotides. Polyacrylamide Gel Electrophoresis (PAGE) is more amenable for longer oligonucleotides, however this method is time and labor intensive and is not suitable for high throughput applications.
Similarly, the presence of dye or fluorescent molecules or modified nucleotides on the oligonucleotides complicates the separation and purification. For example, on some occasions, fluorescent probes are linked to PCR probes that are extended by polymerase, producing a long or full length gene sequence having a fluorescent probe attached. Using conventional purification methods, such labeled oligonucleotides are retained on purification media either too much or too little, and clean separation from unlabeled oligonucleotides is not provided.
Oligonucleotide separation methods are known in the art. For example, Bonn et al. (U.S. Pat. No. 5,585,236) describe a chromatographic method for separating nucleic acids using nonporous polymer beads having an average diameter of 1–100 microns for ion pair reverse phase chromatographic separation when the polymer beads are alkylated with alkyl chains having at least three carbon atoms. In addition, this group reported the advantages of high resolution liquid chromatography of detritylated oligonucleotides on nonporous alkylated polystyrene divinylbenzene copolymers, Huber, D. G., et al., (1993) Anal. Biochem. 212, 351–358.
Similarly, Gjerde et al. (U.S. Pat. No. 6,355,791) describe a nonpolar polymeric chromatographic separation media in the form of beads or monoliths for the separation of mixtures of polynucleotides when the surfaces are substantially free of multivalent cation contamination. The chromatographic separation media are reported to be particularly useful for Matched Ion Polynucleotide Chromatography.
Moore et al., in U.S. patent application No. 2003/0055241, describe a method for purifying oligonucleotides that involves treating a solution with an aggregating agent and a precipitation enhancer, and isolating the purified oligonucleotide.
Separations based on a “fluorous phase” approach, as described in U.S. Pat. No. 6,156,896 to Curran et al., involve the use of silica gel bonded with perfluoroalkyl chains, for purification of highly fluorinated compounds. This technology is commercialized by Fluorous Technologies, Inc. (Pittsburgh, Pa.).
None of the references mentioned above satisfied the need in the art for a fast and convenient method for separating labeled or protected oligonucleotides from unlabeled or deprotected oligonucleotides. In particular, there is a need in the art for a practical and fast method for separating tritylated oligonucleotides from non-tritylated oligonucleotides.